The goal of this project is to design, develop and implement the use of ion-selective, synthetic channel-forming peptides. A primary application for such technology will be the treatment of loss-of-function ion channel diseases such as cystic fibrosis. Epithelial monolayer's act as barriers to the movement of small solute molecules, including both inorganic ions and drugs, between body compartments. Ions cross epithelial apical and basolateral membranes via combination of tightly regulated ion-specific transporters and channels;limited diffusion across tight junctions can occur. Loss-of-function of any channel leads to electrolyte and fluid imbalances that result in morbidity and mortality. For many years, this laboratory has been developing synthetic peptides that form pores with varying degrees of anion conduction and selectivity. These synthetic peptide sequences are distantly related to the pore-forming transmembrane segment (M2) of the spinal cord glycine receptor (GlyR) a1-subunit. We hypothesized that the ideal channel-forming sequence should:1) have high aqueous solubility as a monomer;2)have no detectable antigenicity;3) bind to and then partition rapidly into biological membranes at low solution concentrations;4) undergo supramolecular assembly in the membrane to form pores with high ion throughput;and 5) show physiologically relevant anion selectivity. All of these properties have been achieved with the exception of the final goal, anion selectivity. We are now exploring distinct approaches to raise the permselectivity for Cl- (PCl) relative to either Na+ or K+. Cells treated with our de novopores tolerate them well with the net anion flux controlled by the natural regulation of counter-ion transport. In the Aims we propose to modulate pore-exposed and peptide-peptide interfacial residues that dictate the pore environment, channel geometry, and channel size to generate a series of pores that define a range of perm selectivity's for Cl- relative to monovalentcations, with retention of high anion permeation rates. Specific amino acid replacements will be introduced to modulate pore lining residues with regard to hydrogen bonding capabilities or electrostatic properties. Other replacements will alterpore length and rigidity. The effects of these modifications will be monitored by a combination of electrophysiological and structural (CD &NMR) studies in conjunction with computer modeling.
The bio-based peptide materials described in this proposal are derived from the same biological source and undergo self assemble in both membranes and living cells to form channel pores that display unique biological properties. Producing new peptides with high anion selectivity would have translational promise in the area of channel- replacement therapy for channelopathies such as cystic fibrosis.
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